1. The Field of the Invention
The invention relates to apparatus and methods for fractionating microstructures such as free cells, viruses, macromolecules, or minute particles. More particularly, the present invention relates to apparatus and methods for sorting such microstructures in suspension in a fluid medium, and if desired, for simultaneously viewing individual of those microstructures during the process.
2. Background Art
The sizing, separation, and study of microstructures such as free cells, viruses, macromolecules, and minute particles, are important tools in molecular biology. For example, this fractionation process when applied to DNA molecules is useful in the study of genes and ultimately in planning and the implementation of genetic engineering processes. The fractionation of larger microstructures, such as mammalian cells, promises to afford cell biologists new insight into the functioning of these basic building blocks of living creatures. One method for estimating the size of small DNA molecules is the process of gel electrophoresis.
In gel electrophoresis an agarose gel is spread in a thin layer and allowed to harden into a firm composition. The composition comprises a fine network of fibers retaining therewithin a liquid medium, such as water. The fibers of the agarose gel cross and interact with each other to form a lattice of pores through which molecules smaller than the pores may migrate in the liquid retained in the composition. The size of the pores in the lattice is determined generally by the concentration of the gel used.
Slots are cast in one end of the gel after the gel is hardened, and DNA molecules are placed into the slots. A weak electric field of typically 1-10 volts per centimeter is then generated in the gel by placing the positive pole of an electric power source in one end of the gel and the negative pole of the power source in the opposite end.
In a free solution, the mobility of a DNA molecule is independent of the length of the molecule or of the size of the applied electric field. In a hindered environment, however, aside from the structure of the hindered environment, the mobility of a molecule becomes a function of the length of the molecule and the intensity of the electric field.
The gels used in gel electrophoresis is just such a hindered environment. Molecules are hindered in their migration through the liquid medium in the gel by the lattice structure formed of the fibers in the gel. The molecules nevertheless when inducted by the electric field, move through the gel by migrating through the pores of the lattice structure. Smaller molecules are able to pass through the pores more easily and thus more quickly than are larger molecules. Thus, smaller molecules advance a greater distance through the gel composition in a given amount of time than do larger molecules. The smaller molecules thereby become separated from the larger molecules in the process. In this manner DNA fractionation occurs.
The process has several inherent limitations, however. For example, the pore size in the lattice of gels cannot be accurately measured or depicted. Therefore, the lengths of the molecules migrating through the lattice cannot be accurately measured. It has also been found that DNA molecules larger than 20 megabasepairs in length cannot be accurately fractionated in gels. Apparently, the pore size in the lattice of such materials cannot be increased to permit the fractionation of larger molecules, much less even larger particles, viruses, or free cells.
Further, the lattice structure formed when a gel hardens is not predictable. It is not possible to predict the configuration into which the lattice structure will form or how the pores therein will be positioned, sized, or shaped. The resulting lattice structure is different each time the process is carried out. Therefore, controls and the critical scientific criteria of repeatability cannot be established.
Gel electrophoresis experiments cannot be exactly duplicated in order to predictably repeat previous data. Even if the exact lattice structures formed in one experiment were determinable, the structure could still not be reproduced. Each experiment is different, and the scientific method is seriously slowed.
Also, the lattice structure of a gel is limited to whatever the gel will naturally produce. The general size of the pores can be dictated to a degree by varying the concentration of the gel, but the positioning of the pores and the overall lattice structure cannot be determined or designed. Distinctive lattice structures tailored to specific purposes cannot be created in a gel.
Further, because the lattice structure arrived at depends upon the conditions under which hardening of the gel occurs, the lattice structure even in a single composition need not be uniform throughout.
Another shortcoming of gel electrophoresis is caused by the fact that a gel can only be disposed in a layer that is relatively thick compared to the pores in its lattice structure, or correspondingly to the size of the DNA molecules to be fractionated. Thus, the DNA molecules pass through a gel in several superimposed and intertwined layers. Individual DNA molecules cannot be observed separately from the entire group. Even the most thinly spread gel is too thick to allow an individual DNA molecule moving through the gel to be spatially tracked or isolated from the group of DNA molecules.
The diffusion of long polymers in complex environments where the mobility of the polymer is greatly perturbed is both a challenging statistical physics problem and a problem of great importance in the biological sciences. The length fractionation of charged polymers, such as DNA in gels, is a primary tool of molecular biology. One of the main stumbling blocks to understanding quantitatively the physical principles behind the length-dependent mobility of long polymers in complex environments has, however, been the ill-characterized nature of the hindering environment, the gel.
A known sorting apparatus 20 is illustrated in FIG. 1. Sorting apparatus 20 has utility in fractionating and optionally for simultaneously viewing microstructures, such as free cells, macromolecules, and minute particles in a fluid medium, and doing so as desired in essentially a single layer. Sorting apparatus 20 is comprised of a substrate 22 having a shallow receptacle 24 located on a side 26 thereof. In the embodiment shown, receptacle 24 is recessed in side 26 of substrate 22, although other structures for producing a recess such as receptacle 24 would be workable in the context of the present invention.
Receptacle 24 includes a floor 28 shown to better advantage in FIG. 2 as being bounded by a pair of upstanding opposing side walls 30, 31 and a first end 32 and a second end 34. The height of side walls 30, 31 define a depth of receptacle 24. The depth of receptacle 24 is commensurate with the size of the microstructures to be sorted in sorting apparatus 20. The depth of receptacle 24 is specifically tailored to cause those microstructures in a fluid medium in receptacle 24 to form essentially a single layer. Thus, when the microstructures are caused to migrate in the fluid medium through receptacle 24, the microstructures do so in essentially the single layer. The migration of the microstructures occurs in a migration direction indicated by arrow M defined relative to sorting apparatus 20.
Substrate 22 may be comprised of any type material which can be photolithographically processed. Silicon is preferred, however other materials, such as quartz and sapphire can also be used.
In accordance with one aspect of a known sorting apparatus, such as sorting apparatus 20, capping means are provided for covering receptacle 24 intermediate first end 32 and second end 34 thereof and for affording visual observation of the migration of the microstructures within receptacle 24. As shown by way of example in FIG. 1, a coverslip 36 extends across receptacle 24 in substrate 22 from one of the pair of upstanding opposing side walls 30 to the other of said pair of upstanding opposing side walls 31. The manner by which coverslip 36 is bonded to side 26 of substrate 22 and to the structures therebetween will be discussed in detail subsequently.
According to another aspect of a known sorting apparatus, such as sorting apparatus 20, means are positioned within receptacle 24 for interacting with the microstructures to partially hinder the migration of the microstructures in the migration direction.
As is suggested in the exploded view of FIG. 2, one form of such a means is an array 38 of minute obstacles 39 upstanding from floor 28 of receptacle 24. Obstacles 39 are sized and sized as to advance the particular sorting objective of sorting apparatus 20. The manner of forming obstacles 39 of array 38, as well as another example of another embodiment of obstacles utilizable in such an array, will be discussed in substantial detail below.
Coverslip 36 is so secured to the top of obstacles 39 in array 38 as to preclude migration of microstructures between the obstacles 39 and coverslip 36. Coverslip 36 is optionally transparent, so as to permit visual observation therethrough of the migration of microstructures through array 38. Coverslip 36 may be comprised of any ceramic material. Pyrex is preferred, but other materials, such as quartz and sapphire, for example, may also be used.
In accordance with another aspect of a known sorting apparatus, such as sorting apparatus 20, electric force means is provided for generating an electric field in the fluid medium in receptacle 24. The electric field induces the microstructures to migrate through the fluid medium, either from first end 32 to second end 34 or from second end 34 to first end 32, depending upon the polarity of the electric field and whether the microstructures are positively or negatively charged. Negatively charged microstructures, such as DNA molecules, will be induced to flow toward the positive pole. Positively charged microstructures, such as proteins, will be induced to flow toward the negative pole.
By way of example, a first electrode 40 is shown in FIG. 2 as being located in first end 32 of receptacle 24, and a second electrode 42 is shown as being located in second end 34 of receptacle 24. First electrode 40 and second electrode 42 each comprise a metal strip disposed on floor 28 of receptacle 24.
A battery 44, or other power source is electrically coupled between first and second electrodes 40 and 42, such that first electrode 40 comprises a negative pole and second electrode 42 comprises a positive pole. The electric field generated between first and second electrodes 40 and 42, is non-alternating, but the use of an alternating power source in place of battery 44 would be acceptable.
When DNA is the microstructure being induced to migrate, the electric field intensity in receptacle 24 is in the range of from about 0.1 volt per centimeter to about 20 volts per centimeter.
In FIG. 3, the portion of FIG. 2 encircled by line 3xe2x80x943 is illustrated in an enlarged manner. FIG. 3 illustrates one example of a means for use in a sorting apparatus of the present invention. As shown, array 38 comprises a plurality of obstacles 39 upstanding from floor 28 of receptacle 24. Although FIG. 3 illustrates obstacles 39 as being positioned within array 38 in an ordered and uniform pattern, staggered patterns, or any other predetermined and reproducible pattern, are employable.
FIG. 4 illustrates the various dimension of a typical obstacle 39. The height H of obstacle 39 is measured in a direction normal to floor 28 of receptacle 24. The length L of obstacle 39 is measured in a direction parallel to said migration direction M. The width W of obstacle 39 is measured in a direction normal to the migration direction M. Each of the obstacles 39 are separated from an adjacent obstacle 39 by a predetermined separation distance Sd. The space between adjacent of obstacles 39 is a cross section of array 38 taken normal to floor 28 of receptacle 24 defines a pore 54 of the lattice structure cumulatively produced by obstacles 39 of array 38. For convenience of reference in FIG. 4, such a typical pore 54 has been shaded.
FIG. 4A, a cross-section of two obstacles 39, illustrates in planar view a typical pore 54. Pore 54 compresses the area defined by two obstacles 39 through which a microstructure must pass. Pore 54 is defined by the height H and the separation distance Sd between the obstacles. The desired size of pore 54 is determined by reference to the size of the microstructures to be sorted therethrough. Not only is the pore size of the arrays known, but it is also constant and reproducible.
These dimensions can be changed and designed to be as desired depending upon the type and size of microstructure to be sorted, the design of the array, and the type of obstacles in the array.
For example, the separation distance Sd will vary depending upon whether the migration of microstructures through pores 54 are DNA molecules, viruses and bacterial cells, or mammalian cells. For migration of DNA molecules, the separation distance Sd is within the range of about 0.01 microns to about 20.0 microns. For migration of viruses and bacterial cells, the separation distance Sd is within the range of about 0.01 microns to about 1.0 micron. For migration of mammalian cells, the separation distance is within the range of from about 1.0 micron to about 50.0 microns. It is presently preferred that the separation distance Sd be substantially equal to the radius of gyration of the molecule, the radius of gyration being the distance walking out from the center of the molecule.
Length L also varies depending upon the microstructure to be migrated through array 38 of obstacles 39. In a presently preferred embodiment, the length is generally equal to the separation distance. With regard to height H, the height of obstacles may generally be in the range of from 0.01 microns to about 20.0 microns. For smaller microstructures, the obstacles may have a height in a range from about 0.01 microns to about 0.50 microns. For larger microns, the height may be in the range from about 1.0 micron to about 5.0 microns.
In FIG. 5, another embodiment of an array of obstacles can be seen that is particularly suitable for sorting larger microstructures, such as free cells, viruses, or minute particles. There an array 60 of obstacles in the form of elongated rectangular bunkers 62 is positioned within receptacle 24. Bunkers 62 are comprised of a rectangular shape having opposing sidewalls 64 and a top 66. Bunkers 62 upstand from floor 28 of receptacle 24. bunkers 62 are positioned within columns and rows within receptacle 24. Cells, for example, migrate through the columns and between the rows of bunkers 62 in a migration direction indicated by arrow M. The longitudinal axis of bunkers 62 is disposed in alignment with migration direction M. Channels 68 are formed between rows of bunkers 62 of width W through which the cells migrate. A separation distance Sd, between adjacent rows of bunkers 62 indicates the size of channels 68.
The size and organization of bunkers 62 may vary. Thus, the separation distance Sd may be sized to allow the cells to migrate through channels 68 in essentially a single layer in at least one single file.
The height H of each bunker 62 should also be such as to allow the cells to pass through the bunkers 62 in essentially a single layer. As with sorting apparatus 20, a coverslip 36 is fused to the tops 66 of bunkers 62 to prevent migration of cells between the coverslip and the tops 66 of bunkers 66. This ensures that the cells migrate through the array 60 of bunkers 62 in essentially a single layer.
Bunkers 62 are but examples of obstacles for forming channels 68. Different structures may also be used to simulate channels through which the cells can migrate and be observed.
One known method for making apparatus of the type discussed above involves forming receptacle 24 on one side of substrate 22. Receptacle 24 should be formed of a size such that microstructures migrate in the fluid through receptacle 24 in essentially a single layer. A further step comprises creating array 38 of obstacles 39 within receptacle 24. Each of obstacles 39 have a top 56, sides 57, and a bottom end 58. Obstacles 39 are upstanding from floor 28 of receptacle 24 in a predetermined and reproducible pattern. In one embodiment, the array of obstacles may comprise a plurality of posts.
The creation of obstacles, such as posts, or bunkers, within the receptacle is illustrated in FIGS. 5A-5F. As shown in FIG. 5A, the forming step comprises developing a photosensitive photoresist layer 70 over areas of substrate 22 that are intended to correspond to tops 56 of obstacles 39. This is accomplished by exposing substrate 22 to light through a mask having thereon a corresponding opaque pattern.
The portion of photoresist layer 70 which is exposed to light becomes soluble in a basic developing solution, while the unexposed portion remains on substrate 22 to protect substrate 22. Thus, after development in the developing solution, substrate 22 is left with a pattern of photoresist layer 70 that is identical to the opaque pattern of the mask. FIG. 5B illustrates substrate 22 with photoresist layer 70 thereon after exposure to light and development in solution.
The next step comprises etching substrate 22 such that the areas of substrate 22 unshielded by photoresist layer 70 are exposed to the etching, thereby forming receptacle 24. The array 38 of obstacles 39 upstanding within the etched receptacle 24 is formed by the portions of substrate 22 shielded by photoresist layer 70. FIG. 5C illustrates formation of receptacle 24 and the obstacles 39.
As can be seen in FIG. 5C, as the substrate 22 is etched, the photoresist layer 70 is also etched, but at a slower rate. FIG. 5C illustrates the receptacle 24 half formed, and photoresist layer 70 partially etched away. If, for example, the photoresist layer is etched at a rate {fraction (1/10)} the rate that substrate 22 is etched, the resulting receptacle can at most have a depth ten times the thickness of the photoresist layer. The thickness of photoresist layer 70 must therefore be chosen accordingly.
The etching process can be terminated at any time when the desired depth of the receptacle is reached. As illustrated in FIG. 5D, there may be some photoresist layer 70 still present on substrate 22 when the etching is terminated. If so, the next step is then dissolving photoresist layer 70 from substrate 22. This step leaves a clean substrate 22 as shown in FIG. 5E.
Etching may be effected by many methods. In the preferred embodiment, ion milling is used such that an overhead ion beam is used to etch the substrate 22 and photoresist layer 70. Other methods of etching, such as chemical etching, are also within the scope of the present invention.
The important step of fusing coverslip 36 to substrate 22 is illustrated in FIG. 5F as comprises positioning coverslip 36 over array 38 of obstacles 39, such that coverslip 36 is in contact with each of obstacles 39, and then applying an electric field between coverslip 36 and each of obstacles 39. The coverslip 36 is held with a negative potential. The obstacles 39 are held at a positive potential. Ions are thereby induced to migrate there between to create a bond between coverslip 36 and each of obstacles 39 at all areas of contact. The process of this step is referred to as field assisted fusion.
The voltage used to fuse coverslip 36 to the substrate 22 is preferably about 1 kilovolt but can be within the range of from 200 volts to about 2000 volts. The time for fusion is about 30 minutes at a temperature of about 400xc2x0 C. The temperature can also range from about 300xc2x0 C. to about 600xc2x0 C., with 400xc2x0 C. being the preferred temperature. In one embodiment, the coverslip comprises a Pyrex material. For example, sapphire, and quartz are materials which may also be used for the coverslip. Any ceramic material or an opaque material may also serve.
In the context of using field assisted fusion to secure the coverslip and substrate, it is advisable that the material used for coverslip 36 have substantially the same coefficient of thermal expansion as substrate 22. Otherwise, at the high temperature of fusion, the coverslip 36 and the substrate 22 will expand at different rates and a seal between the two would be difficult or impossible to accomplish.
Successful fusion can be tested by injecting a fluorescent fluid into the apparatus. A completely fused coverslip will not allow passage of any fluorescent fluid between coverslip 36 and obstacles 39.
The method of making the apparatus disclosed above, by involving Pyrex-silicon based anodic bonding to enclose the microstructure, entails high temperatures and high voltage conditions, either of which can damage components. The binding of the cover of the apparatus to the array thereof renders the resulting apparatus usable only once.
As the sealing of the structure is irreversible, access is precluded to sorted microstructures inside the structure, unless the device is destructively disassembled.
It is an object of the present invention to provide further methods and apparatus for fractionation of microstructures.
Another object of the present invention is to provide improved microstructure sorting devices and associated methods.
An additional object of the present invention to provide a method for accessing sorted microstructures or particles inside a microfabricated microstructure sorting device, thereby to permit further analysis of those sorted microstructures or particles.
An additional object of the present invention is to provide further methods for making microstructure sorting devices.
Additional objects and advantages of the invention are set forth hereinbelow in the detailed described or will be appreciated by the practice of the invention.
To achieve the foregoing objects, and in accordance with the invention as embodied and broadly described herein, the present invention is directed to a method for hermetically and reversibly sealing a microfabricated sorting array. The cover and the microfabricated sorting array are nondestructively separable, allowing access to any sorted microstructures or particles therein and allowing the reuse after cleaning of the sorting array.
The present invention utilizes a silicone elastomer in various combinations with rigid materials, such as silicon. A microfabricated sorting array may be constructed photolithographically from a material such as silicon, or may be formed from an elastomeric material as, for example, by casting from a correspondingly configured microfabricated mold of silicon or of elastomer. Reversible sealing allows for access to the fractionated microstructures within the array.
Thus provided is an apparatus for sorting microstructures in a fluid medium. The apparatus includes a substrate having a floor bound on opposite sides by first and second side walls. The floor and the first and second side walls define a receptacle. Means are positioned within the receptacle for showing the rate of migration of microstructures within the receptacle. A cover that seals said receptacle and contacts the ends of the means opposite from the floor of the receptacle, is selectively separable therefrom. One of the cover or the substrate is comprised of an elastomer. The other may be comprised of silicon, quartz, sapphire, or even an elastomer.
In alternative embodiments of the invention, an apparatus for sorting microstructures in a fluid medium includes a rigid support backing for either of the substrate or the cover that is comprised of an elastomer.
Also disclosed according to the teachings of the present invention is a method of manufacturing an apparatus for sorting microstructures in a fluid medium. The method includes a step of providing a substrate having a floor bounded by opposed first and second side walls. The floor in combination with the first and second side walls defines a receptacle. In the receptacle, means are provided for slowing the rate of migration of microstructures through the receptacle. The method further comprises the steps of forming a cover and removably engaging the cover with the receptacle and with the ends of the means opposite from the floor of the receptacle.
The present invention thus provides methods and apparatus that facilitate the fractionation and study of many types of microstructures. The present invention allows the successful fractionation of DNA molecules of chromosomal length in quantities so small as to isolate single of those molecules. The present invention also facilitates the fractionation of larger microstructures, such as red blood cells.
The fractionation of other macromolecules and microstructures, such as proteins, polymers, viruses, other cells, and minute particles, is also considered to be within the scope of the present invention, however.